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Ian Brierley

Publications and source records attributed to Ian Brierley.

11 recordsLinked to original sources
PubMed2026

A protein-dependent riboswitch activates ribosomal frameshifting in cardioviruses.

Programmed -1 ribosomal frameshifting (PRF) is a translational control mechanism used by RNA viruses to regulate the relative abundance of proteins encoded in different reading frames. Cardioviruses exhibit the highest known PRF efficiency, with ∼85% of ribosomes shifting into the -1 frame. This unusual event requires an interaction between the viral 2A protein and a stimulatory element in the RNA genome, but the basis for protein dependence is unclear. To address this, here we investigate the structure and dynamics of the PRF signal in Theiler's murine encephalitis virus (TMEV). By combining X-ray crystallography, small-angle X-ray scattering (SAXS), and single-molecule fluorescence resonance energy transfer (smFRET), we show that 2A binding switches the RNA from a stem-loop conformation into a pseudoknot, and we demonstrate that pseudoknot formation is essential for efficient PRF in vitro and in cells. Together, these findings illustrate how the cardiovirus PRF element behaves as a protein-dependent riboswitch, defining the molecular mechanism by which frameshifting is conditionally activated.

Frameshifting, Ribosomal↗
PubMed2007

Structure-function analysis of the ribosomal frameshifting signal of two human immunodeficiency virus type 1 isolates with increased resistance to viral protease inhibitors.

Expression of the pol-encoded proteins of human immunodeficiency virus type 1 (HIV-1) requires a programmed -1 ribosomal frameshift at the junction of the gag and pol coding sequences. Frameshifting takes place at a heptanucleotide slippery sequence, UUUUUUA, and is enhanced by a stimulatory RNA structure located immediately downstream. In patients undergoing viral protease (PR) inhibitor therapy, a p1/p6(gag) L449F cleavage site (CS) mutation is often observed in resistant isolates and frequently generates, at the nucleotide sequence level, a homopolymeric and potentially slippery sequence (UUUUCUU to UUUUUUU). The mutation is located within the stimulatory RNA downstream of the authentic slippery sequence and could act to augment levels of pol-encoded enzymes to counteract the PR deficit. Here, RNA secondary structure probing was employed to investigate the structure of a CS-containing frameshift signal, and the effect of this mutation on ribosomal frameshift efficiency in vitro and in tissue culture cells was determined. A second mutation, a GGG insertion in the loop of the stimulatory RNA that could conceivably lead to resistance by enhancing the activity of the structure, was also tested. It was found, however, that the CS and GGG mutations had only a very modest effect on the structure and activity of the HIV-1 frameshift signal. Thus the increased resistance to viral protease inhibitors seen with HIV-1 isolates containing mutations in the frameshifting signal is unlikely to be accounted for solely by enhancement of frameshift efficiency.

Drug Resistance, Viral↗
PubMed2006

Specific binding of Bluetongue virus NS2 to different viral plus-strand RNAs.

The Reoviridae have double-stranded RNA genomes of 10-12 segments, each in a single copy in the mature virion. The basis of genome segment sorting during virus assembly that ensures each virus particle contains the complete viral genome is unresolved. Bluetongue virus (BTV) NS2 is a single-stranded RNA-binding protein that forms inclusion bodies in infected cells. Here, we demonstrate that the specific interaction between NS2 and a stem-loop structure present in BTV S10 RNA, and phylogenetically conserved in other BTV serotypes, is abolished by mutations predicted to disrupt the structure. Subsequently, we mapped RNA regions in three other genomic segments of BTV that are bound preferentially by NS2. However, structure probing of these RNAs did not reveal secondary structure motifs that obviously resembled the stem-loop implicated in the NS2-S10 interaction. In addition, the specific binding by NS2 to two different viral RNAs was found to occur independently. Together, these data support the hypothesis that the recognition by NS2 of different RNA structures may be the basis for discrimination between viral RNAs during virus assembly.

Animals↗
PubMed2006

A mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting.

The triplet-based genetic code requires that translating ribosomes maintain the reading frame of a messenger RNA faithfully to ensure correct protein synthesis. However, in programmed -1 ribosomal frameshifting, a specific subversion of frame maintenance takes place, wherein the ribosome is forced to shift one nucleotide backwards into an overlapping reading frame and to translate an entirely new sequence of amino acids. This process is indispensable in the replication of numerous viral pathogens, including HIV and the coronavirus associated with severe acute respiratory syndrome, and is also exploited in the expression of several cellular genes. Frameshifting is promoted by an mRNA signal composed of two essential elements: a heptanucleotide 'slippery' sequence and an adjacent mRNA secondary structure, most often an mRNA pseudoknot. How these components operate together to manipulate the ribosome is unknown. Here we describe the observation of a ribosome-mRNA pseudoknot complex that is stalled in the process of -1 frameshifting. Cryoelectron microscopic imaging of purified mammalian 80S ribosomes from rabbit reticulocytes paused at a coronavirus pseudoknot reveals an intermediate of the frameshifting process. From this it can be seen how the pseudoknot interacts with the ribosome to block the mRNA entrance channel, compromising the translocation process and leading to a spring-like deformation of the P-site transfer RNA. In addition, we identify movements of the likely eukaryotic ribosomal helicase and confirm a direct interaction between the translocase eEF2 and the P-site tRNA. Together, the structural changes provide a mechanical explanation of how the pseudoknot manipulates the ribosome into a different reading frame.

Animals↗
PubMed2005

Programmed ribosomal frameshifting in HIV-1 and the SARS-CoV.

Ribosomal frameshifting is a mechanism of gene expression used by several RNA viruses to express replicase enzymes. This article focuses on frameshifting in two human pathogens, the retrovirus human immunodeficiency virus type 1 (HIV-1) and the coronavirus responsible for severe acute respiratory syndrome (SARS). The nature of the frameshift signals of HIV-1 and the SARS-CoV will be described and the impact of this knowledge on models of frameshifting will be considered. The role of frameshifting in the replication cycle of the two pathogens and potential antiviral therapies targeting frameshifting will also be discussed.

Base Sequence↗
PubMed2005

Characterization of the frameshift signal of Edr, a mammalian example of programmed -1 ribosomal frameshifting.

The ribosomal frameshifting signal of the mouse embryonal carcinoma differentiation regulated (Edr) gene represents the sole documented example of programmed -1 frameshifting in mammalian cellular genes [Shigemoto,K., Brennan,J., Walls,E,. Watson,C.J., Stott,D., Rigby,P.W. and Reith,A.D. (2001), Nucleic Acids Res., 29, 4079-4088]. Here, we have employed site-directed mutagenesis and RNA structure probing to characterize the Edr signal. We began by confirming the functionality and magnitude of the signal and the role of a GGGAAAC motif as the slippery sequence. Subsequently, we derived a model of the Edr stimulatory RNA and assessed its similarity to those stimulatory RNAs found at viral frameshift sites. We found that the structure is an RNA pseudoknot possessing features typical of retroviral frameshifter pseudoknots. From these experiments, we conclude that the Edr signal and by inference, the human orthologue PEG10, do not represent a novel 'cellular class' of programmed -1 ribosomal frameshift signal, but rather are similar to viral examples, albeit with some interesting features. The similarity to viral frameshift signals may complicate the design of antiviral therapies that target the frameshift process.

Animals↗
PubMed2004

Reprogrammed genetic decoding in cellular gene expression.

Reprogrammed genetic decoding signals in mRNAs productively overwrite the normal decoding rules of translation. These "recoding" signals are associated with sites of programmed ribosomal frameshifting, hopping, termination codon suppression, and the incorporation of the unusual amino acids selenocysteine and pyrrolysine. This review summarizes current knowledge of the structure and function of recoding signals in cellular genes, the biological importance of recoding in gene regulation, and ways to identify new recoded genes.

Animals↗
PubMed2003

Prokaryotic-style frameshifting in a plant translation system: conservation of an unusual single-tRNA slippage event.

Ribosomal frameshifting signals are found in mobile genetic elements, viruses and cellular genes of prokaryotes and eukaryotes. Typically they comprise a slippery sequence, X XXY YYZ, where the frameshift occurs, and a stimulatory mRNA element. Here we studied the influence of host translational environment and the identity of slippery sequence-decoding tRNAs on the frameshift mechanism. By expressing candidate signals in Escherichia coli, and in wheatgerm extracts depleted of endogenous tRNAs and supplemented with prokaryotic or eukaryotic tRNA populations, we show that when decoding AAG in the ribosomal A-site, E.coli tRNA(Lys) promotes a highly unusual single-tRNA slippage event in both prokaryotic and eukaryotic ribosomes. This event does not appear to require slippage of the adjacent P-site tRNA, although its identity is influential. Conversely, asparaginyl-tRNA promoted a dual slippage event in either system. Thus, the tRNAs themselves are the main determinants in the selection of single- or dual-tRNA slippage mechanisms. We also show for the first time that prokaryotic tRNA(Asn) is not inherently 'unslippery' and induces efficient frameshifting when in the context of a eukaryotic translation system.

Base Sequence↗
PubMed2003

Sequence specificity in the interaction of Bluetongue virus non-structural protein 2 (NS2) with viral RNA.

The non-structural protein NS2 of Bluetongue virus (BTV) is synthesized abundantly in virus-infected cells and has been suggested to be involved in virus replication. The protein, with a high content of charged residues, possesses a strong affinity for single-stranded RNA species but, to date, all studies have failed to identify any specificity in the NS2-RNA interaction. In this report, we have examined, through RNA binding assays using highly purified NS2, the specificity of interaction with different single-stranded RNA (ssRNA) species in the presence of appropriate competitors. The data obtained show that NS2 indeed has a preference for BTV ssRNA over nonspecific RNA species and that NS2 recognizes a specific region within the BTV10 segment S10. The secondary structure of this region was determined and found to be a hairpin-loop with substructures within the loop. Modification-inhibition experiments highlighted two regions within this structure that were protected from ribonuclease cleavage in the presence of NS2. Overall, these data imply that a function of NS2 may be to recruit virus messenger RNAs (that also act as templates for synthesis of genomic RNAs) selectively from other RNA species within the infected cytosol of the cell during virus replication.

Animals↗
PubMed2003

An internal ribosome entry site directs translation of the murine gammaherpesvirus 68 MK3 open reading frame.

The gammaherpesviruses characteristically drive the proliferation of latently infected lymphocytes. The murine gammaherpesvirus 68 (MHV-68) MK3 protein contributes to this process in vivo by evading CD8(+)-T-cell recognition during latency, as well as during lytic infection. We analyzed some of the molecular mechanisms that control MK3 expression. No dedicated MK3 mRNA was detected. Instead, the MK3 open reading frame (ORF) was transcribed as part of a bicistronic mRNA, downstream of a previously unidentified ORF, 13M. The 13M/MK3 promoter appeared to extend approximately 1 kb 5' of the transcription start site and included elements both dependent on and independent of the ORF50 lytic transactivator. MK3 was translated from the bicistronic transcript by virtue of an internal ribosome entry site (IRES) element. RNA structure mapping identified two stem-loops between 13M and MK3 that were sufficient for IRES activity in a bicistronic reporter plasmid and a third stem-loop just within the MK3 coding sequence, with a subtler, perhaps regulatory role. Overall, translation of the MHV-68 MK3 bore a striking resemblance to that of the Kaposi's sarcoma-associated herpesvirus vFLIP, suggesting that IRES elements are a common theme of latent gammaherpesvirus immune evasion in proliferating cells.

5' Untranslated Regions↗